Biologically functional, non-covalently linked complexes of cytochrome c are generated from several combinations of overlapping and non-overlapping fragments. The use of fast kinetic techniques to study the dynamic events leading to the formation of an ordered structure suggested that the process occurs in at least two distinct phase. We observed an initial, second order reaction in which the fragments combined to form a product have a structure reasonably similar to native cytochrome c. Additional evidence indicated that achievement of a native-like ligation state of the heme iron was a distinct kinetic event occurring after the second order reaction. This proposition has been tested by using chemically modified derivatives of the original fragments. These modifications prevent formation of a ligation state identical to that of native cytochrome c. In one instance the iron atom is removed from the heme moiety and in the other the methionine residues are converted to methionine sulfoxide. These derivatives exhibit second order kinetic behavior nearly identical to that of the original fragments. This provides strong support for the idea that formation of the native-like structure occurs in at least two kinetically distinct phases.